Fourier Transform Infrared Difference Spectroscopy of Secondary Quinone Acceptor Photoreduction in Proton Transfer Mutants of Rhodobacter sphaeroides

Nabedryk, E.; Breton, Jacques; Hienerwadel, Rainer; Fogel, C.; Mäntele, Werner; Ml, Paddock; My, Okamura

doi:10.1021/bi00045a013

Cited by 102 publications

(203 citation statements)

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“…For a given sample, these measurements were repeated over ∼30 h. Spectra are an average of two to three samples. , C-O-CH 3 at 1265 and 1290 cm -1 ), and the protein at 1728, 1685, 1651, 1641, 1554, 1537, 1527, and 1514 cm -1 (14,42,44). Among the series of L209 mutants, the Q B -/Q B spectrum of Thr-L209 RCs (Figure 2b) is the only one that is close to that of native RCs.…”

Section: Methodsmentioning

confidence: 87%

“…Both spectra of Phe-L209 ( Figure 2d In all the mutant spectra (Figure 2), contributions from the methoxy groups of Q B are consistently detected at ∼1290 and 1265 cm (14,(19)(20)(21). , and 1400 and 1200 cm -1 which are not affected by the binding of labeled quinone, correspond mostly to the absorption of protein and unlabeled quinone (methyl, methoxy, and isoprenoid chain) groups.…”

Section: Q Bmentioning

confidence: 88%

“…In addition to the determination of the bonding interactions of the neutral Q B , light-induced FTIR difference spectroscopy provides means to explore the protein structural rearrangements (backbone and side chains), the changes of protonation state of carboxylic acids, and the semiquinone-protein interactions (14,42,44,45). In this report, the effect of substituting Pro-L209 with Thr, Glu, Phe, and Tyr in RCs from Rb.…”

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confidence: 93%

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Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

et al. 2003

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The effect of substituting Pro-L209 with Tyr, Phe, Glu, and Thr in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was investigated by monitoring the light-induced FTIR absorption changes associated with the photoreduction of the secondary quinone Q B . Pro-L209 is close to a chain of ordered water molecules connecting Q B to the bulk phase. In wild-type RCs, two distinct main Q B binding sites (distal and proximal to the non-heme iron) have been described in the literature. The X-ray structures of the mutant RCs Pro-L209 f Tyr, Pro-L209 f Phe, and Pro-L209 f Glu have revealed that Q B occupies a proximal, intermediate, and distal position, respectively [Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G. (2001) Biochemistry 40, 4253-4260]. FTIR absorption changes associated with the reduction of Q B in Pro-L209 f Phe RCs reconstituted with 13 C-labeled ubiquinone show a highly specific IR fingerprint for the CdO and CdC modes of Q B upon selective labeling at C 1 or C 4 . This IR fingerprint is similar to those of wild-type RCs and the Pro-L209 f Tyr mutant [Breton, J., Boullais, C., Mioskowski, C., Sebban, P., Baciou, L., and Nabedryk, E. (2002) Biochemistry 41, 12921-12927], demonstrating that equivalent interactions occur between neutral Q B and the protein in wild-type and mutant RCs. It is concluded that in all RCs, neutral Q B in its functional state occupies a unique binding site which is favored to be the proximal site. This result contrasts with the multiple Q B binding sites found in crystal structures. With respect to wild-type RCs, the largest FTIR spectral changes upon Q B -formation are observed for the Phe-L209 and Tyr-L209 mutants which undergo similar protein structural changes and perturbations of the semiquinone modes. Smaller changes are observed for the Glu-L209 mutant, while the vibrational properties of the Thr-L209 mutant are essentially the same as those for native RCs.The reaction center (RC) 1 from the photosynthetic purple bacterium Rhodobacter (Rb.) sphaeroides is a transmembrane protein complex that couples the light-induced transfer of two electrons and two protons to the secondary quinone Q B to form the quinol which then leaves the RC to be oxidized by another membrane protein complex, the cytochrome bc 1 complex (1). The resulting transmembrane proton gradient drives ATP synthesis. Since Q B is located in the interior of the protein, the protons required to stabilize the reduced quinone must be conducted through the protein from the cytoplasmic surface to the Q B binding site. In the highresolution structures of bacterial RCs, chains of protonatable amino acid residues and ordered water molecules that connect Q B to the bulk phase have been identified (2-6) and are presumably used for the uptake, transport, and delivery of protons to Q B . A number of spectroscopic studies of sitedirected mutant RCs from Rb. sphaeroides (for reviews, see refs 1, 7, and 8) have led to the identification of specific ionizable side chains, such as th...

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Section: Methodsmentioning

confidence: 87%

Section: Q Bmentioning

confidence: 88%

mentioning

confidence: 93%

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Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

et al. 2003

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“…However, at pH 7.8, in the presence of 1 mM Cd 2+ concentration, k AB (1) is much more reduced in the L209PY mutant (∼120 s -1 ; ∼75 times) than in the WT (∼700 s -1 ; ∼10 times). k AB (1) is mainly governed by the protonation state of L212Glu (17,(28)(29)(30). We have previously suggested that the L209PY mutantion may have disorganized the hydrogen-bond network involving water molecules and protonatable groups, which may serve as proton provision for L212Glu (24).…”

Section: Discussionmentioning

confidence: 99%

Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

et al. 2002

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In bacterial reaction center of Rhodobacter sphaeroides, Cd 2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (k AB (1)) and second flash (k AB (2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogenbond networks involving water molecules. We show that (1) the combined effects of Cd 2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd 2+ ] titration curves of k AB (2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S. A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd 2+ binds; (2) in the L209PY mutant, the pH titration curves of k AB (1), k AB (2), and k H + , the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 µM Cd 2+

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“…The partial protonation events that occur on Q A Ϫ and Q B Ϫ formations have been studied by spectroscopic techniques by using site-directed mutagenesis (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and by numerical methods (23)(24)(25)(26)(27)(28)(29)(30). There is a general agreement that the major response of the protein to the Q B Ϫ formation is the change of the ionization state of acidic residues situated in the Q B environment.…”

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confidence: 99%

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

Tandori

Maróti

Alexov

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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T he biological role of bacterial reaction center (RC) membrane proteins is to convert light energy into chemical free energy. The sequential absorption of two photons by the system results into the production of the doubly reduced and doubly protonated form of the ultimate electron acceptor of the complex, a ubiquinone (Q B ). The formed Q B H 2 molecule then delivers its reducing power to the cytochrome bc1 complex, resulting in the release of protons on the periplasmic side of the membrane. The resulting transmembrane proton gradient drives ATP synthesis through the ATP synthase. The reduction of Q B coupled to the uptake of protons from the bulk is an important step shared by many systems involved either in photosynthesis or in respiratory chains (1).The three-dimensional structure of the reaction center from the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is known at atomic resolution (2-4). Three subunits with a total molecular weight of about 100 kDa compose these RCs. The transmembrane L and M subunits carry the nine pigments and cofactors: four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones 10, and one non-heme iron atom. The third subunit, H, caps the reaction center on the cytoplasmic side of the membrane. The initial photochemical event induced by the absorption of a photon is the creation of the singlet excited state of a dimer of bacteriochlorophylls (P3P*), which constitutes the primary electron donor. P* is a strong reducing species that initiates the electron transfer reaction through the protein. In about 200 ps, the charge separation occurs between P and the first quinone electron acceptor, Q A , situated on the cytoplasmic side of the complex. The electron is then transferred from Q A Ϫ to a secondary quinone Q B within 10-100 s (5-7). Both Q A and Q B are deeply buried within the reaction center protein. The role of the protein in stabilizing the redox species is essential to ensure high forward electron transfer rates and to prevent charge recombinations to occur. Although chemically identical, Q A and Q B behave differently. Q A , bound to the M subunit in a relatively hydrophobic pocket, functions as one electron acceptor and is never protonated. At variance, Q B , bound to the L subunit, is surrounded by charged and polar residues and behaves as a two-electron gate, accepting sequentially two electrons from Q A and two protons from the cytoplasm. In chromatophores, the semiquinone Q B Ϫ can bind a proton below pH 6.8 (8). However, in isolated RCs, the semiquinones are not directly protonated but induce the shift of the pKas of ionizable interacting residues, which results in substoichiometric proton uptake by the protein (9, 10). The proton uptake may occur through a number of water molecules and protonatable amino acid residues situated between Q B and the cytoplasmic surface. Of main interest is to identify the dynamical and structural role of the protein that contributes to the stability of the Q A Ϫ and Q B Ϫ states and to the energetic and functiona...

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Fourier Transform Infrared Difference Spectroscopy of Secondary Quinone Acceptor Photoreduction in Proton Transfer Mutants of Rhodobacter sphaeroides

Cited by 102 publications

References 36 publications

Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

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Fourier Transform Infrared Difference Spectroscopy of Secondary Quinone Acceptor Photoreduction in Proton Transfer Mutants of Rhodobacter sphaeroides

Cited by 102 publications

References 36 publications

Quinone (QB) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Quinone (QB) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Effect of Binding of Cd2+ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides

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Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Quinone (Q_B) Binding Site and Protein Stuctural Changes in Photosynthetic Reaction Center Mutants at Pro-L209 Revealed by Vibrational Spectroscopy

Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways